Polypeptides Having Cellobiohydrolase Activity And Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/405,988filed on Feb. 27, 2012, which is a divisional of U.S. application Ser.No. 12/680,433 filed on Mar. 26, 2010, now U.S. Pat. No. 8,124,394,which is a 35 U.S.C. §371 national application of PCT/US2008/077864filed on Sep. 26, 2008 and claims priority from U.S. ProvisonalApplication No. 60/976,207 filed on Sep. 28, 2007, which applicationsare incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingcellobiohydrolase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose can be easily fermented by yeast into ethanol.

WO 2004/056981 discloses cellobiohydrolases from Chaetomium thermophilumand Myceliophthora thermophila. Gusakov et al., 2007, BiotechnologyBioengineering 97: 1028-1038, describe a cellobiohydrolase isolated fromChrysosporium lucknowense.

The present invention relates to polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence having at least 80%identity to the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 80% identity to the mature polypeptide codingsequence of SEQ ID NO: 1; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more (several) amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having cellobiohydrolase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence having at least 80% identity to the mature polypeptide of SEQID NO: 2;

(b) a polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of(i) or (ii);

(c) a polynucleotide comprising a nucleotide sequence having at least80% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of themature polypeptide of SEQ ID NO: 2.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingcellobiohydrolase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of a polynucleotide of thepresent invention. The present also relates to such a double-strandedinhibitory RNA (dsRNA) molecule, wherein optionally the dsRNA is a siRNAor a miRNA molecule.

The present invention also relates to methods of using the polypeptideshaving cellobiohydrolase in detergents and in the conversion ofcellulose to glucose and various substances.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding such a polypeptide having cellobiohydrolaseactivity.

The present invention also relates to methods of producing such apolypeptide having cellobiohydrolase, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingsuch a polypeptide having cellobiohydrolase activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene isforeign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of a Myceliophthora thermophila CBS 202.75 cellobiohydrolase(SEQ ID NOs: 1 and 2, respectively).

FIG. 2 shows a restriction map of pSMai180.

FIG. 3 shows a restriction map of pSMai182.

DEFINITIONS

Cellobiohydrolase activity: The term “cellobiohydrolase activity” isdefined herein as a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91)activity that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkagesin cellulose, cellotetriose, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing end ofthe chain. For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBSLetters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters,187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. Inthe present invention, the Lever et al. method can be employed to assesshydrolysis of cellulose in corn stover, while the methods of vanTilbeurgh et al. and Tomme et al. can be used to determinecellobiohydrolase activity on a fluorescent disaccharide derivative.

Endoglucanase activity: The term “endoglucanase activity” is definedherein as an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C.3.2.1.4) that catalyses endohydrolysis of 1,4-beta-D-glycosidic linkagesin cellulose, cellulose derivatives (such as carboxy methyl celluloseand hydroxy ethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Beta-glucosidase activity: The term “beta-glucosidase activity” isdefined herein as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21)activity that catalyzes the hydrolysis of terminal non-reducingbeta-D-glucose residues with the release of beta-D-glucose. Cellobiaseis synonymous with beta-glucosidase. For purposes of the presentinvention, beta-glucosidase activity is determined at 25° C. using 1 mM4-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate pH 4.8. One unit of beta-glucosidase activity is defined as 1.0pmole of 4-nitrophenol produced per minute at 25° C., pH 4.8.

Family 6 or Family GH6 or Cel6: The term “Family 6” or “Family GH6” or“Cel6” is defined herein as a polypeptide falling into the glycosidehydrolase Family 6 according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696. According to such a classification, SEQ ID NO: 2 or the maturepolypeptide thereof belongs to Family 6 and is predicted to be acellobiohydrolase II.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having cellobiohydrolase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc. In a preferred aspect, the mature polypeptide isamino acids 18 to 482 of SEQ ID NO: 2 based on the SignalP softwareprogram (Nielsen et al., 1997, Protein Engineering 10:1-6) that predictsamino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having cellobiohydrolase activity. In a preferredaspect, the mature polypeptide coding sequence is nucleotides 52 to 1809of SEQ ID NO: 1 based on the SignalP software program (Nielsen et al.,1997, Protein Engineering 10:1-6) that predicts nucleotides 1 to 51encode a signal peptide.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the −nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the −nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein that gives an E value (or expectancy score) of lessthan 0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Myceliophthora thermophila cellobiohydrolase of SEQ ID NO: 2 orthe mature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide of SEQ IDNO: 2; or a homologous sequence thereof; wherein the fragment hascellobiohydrolase activity. In a preferred aspect, a fragment containsat least 415 amino acid residues, more preferably at least 435 aminoacid residues, and most preferably at least 455 amino acid residues, ofthe mature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1;or a homologous sequence thereof; wherein the subsequence encodes apolypeptide fragment having cellobiohydrolase activity. In a preferredaspect, a subsequence contains at least 1245 nucleotides, morepreferably at least 1305 nucleotides, and most preferably at least 1365nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 ora homologous sequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2; or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be a substitution, a deletion and/or an insertion of one or more(several) amino acids as well as replacements of one or more (several)amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having cellobiohydrolase activity produced by anorganism expressing a modified polynucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1; or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the polynucleotide sequence disclosed inSEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingCellobiohydrolase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence having a degree ofidentity to the mature polypeptide of SEQ ID NO: 2 of preferably atleast 60%, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99%, which have cellobiohydrolaseactivity (hereinafter “homologous polypeptides”). In a preferred aspect,the homologous polypeptides have an amino acid sequence that differs byten amino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having cellobiohydrolase activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 18 to 482 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseactivity. In another preferred aspect, the polypeptide comprises aminoacids 18 to 482 of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having cellobiohydrolaseactivity. In another preferred aspect, the polypeptide consists of theamino acid sequence of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of the mature polypeptide of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of amino acids 18 to482 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereofhaving cellobiohydrolase activity. In another preferred aspect, thepolypeptide consists of amino acids 18 to 482 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having cellobiohydrolase activity that are encoded bypolynucleotides that hybridize under preferably very low stringencyconditions, more preferably low stringency conditions, more preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1,(iii) a subsequence of (i) or (ii), or (iv) a full-length complementarystrand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, ColdSpring Harbor, N.Y.). A subsequence of the mature polypeptide codingsequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides orpreferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment having cellobiohydrolaseactivity. In a preferred aspect, the complementary strand is thefull-length complementary strand of the mature polypeptide codingsequence of SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; aswell as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof;may be used to design nucleic acid probes to identify and clone DNAencoding polypeptides having cellobiohydrolase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes that are preferably at least 600 nucleotides, morepreferably at least 700 nucleotides, even more preferably at least 800nucleotides, or most preferably at least 900 nucleotides in length. BothDNA and RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having cellobiohydrolase activity.Genomic or other DNA from such other strains may be separated by agaroseor polyacrylamide gel electrophoresis, or other separation techniques.DNA from the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1,or a subsequence thereof, the carrier material is preferably used in aSouthern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1;the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1; its full-length complementary strand; or a subsequencethereof; under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 52 to 1809 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencethat encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pSMai182 which is contained in E. coliNRRL B-50059, wherein the polynucleotide sequence thereof encodes apolypeptide having cellobiohydrolase activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pSMai182 which is contained in E. coli NRRLB-50059.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having cellobiohydrolase activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 of preferably at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, or at least 99%,which encode an active polypeptide. See polynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or more (orseveral) amino acids of the mature polypeptide of SEQ ID NO: 2, or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,cellobiohydrolase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides that arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 18 to 482 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Cellobiohydrolase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide having cellobiohydrolase activity of the present inventionmay be a bacterial polypeptide. For example, the polypeptide may be agram positive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide havingcellobiohydrolase activity, or a Gram negative bacterial polypeptidesuch as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having cellobiohydrolase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellobiohydrolase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide havingcellobiohydrolase activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellobiohydrolase activity.

A polypeptide having cellobiohydrolase activity of the present inventionmay also be a fungal polypeptide, and more preferably a yeastpolypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having cellobiohydrolaseactivity; or more preferably a filamentous fungal polypeptide such as anAcremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide having cellobiohydrolase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide havingcellobiohydrolase activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride polypeptide having cellobiohydrolase activity.

In another preferred aspect, the polypeptide is a Myceliophthorathermophila, Myceliophthora fergusii, Myceliophthora hinnulea,Myceliophthora histoplasmoides, Myceliophthora indica, Myceliophthoralutea, Myceliophthora sulphurea, Myceliophthora thermophila, orMyceliophthora vellerea polypeptide.

In a more preferred aspect, the polypeptide is a Myceliophthorathermophila polypeptide having cellobiohydrolase activity. In a mostpreferred aspect, the polypeptide is a Myceliophthora thermophila CBS202.75 polypeptide having cellobiohydrolase activity, e.g., thepolypeptide comprising the mature polypeptide of SEQ ID NO: 2.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotideencoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having cellobiohydrolase activity from the fusion protein.Examples of cleavage sites include, but are not limited to, a Kex2 sitethat encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that encodepolypeptides having cellobiohydrolase activity of the present invention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pSMai182which is contained in E. coli NRRL B-50059. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 52 to 1809 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pSMai182 which is contained in E. coli NRRLB-50059. The present invention also encompasses nucleotide sequencesthat encode polypeptides comprising or consisting of the amino acidsequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differfrom SEQ ID NO: 1 or the mature polypeptide coding sequence thereof byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ IDNO: 2 that have cellobiohydrolase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Myceliophthora, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 ofpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode an activepolypeptide.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the mature polypeptide coding sequenceof SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions that do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionsthat may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for cellobiohydrolase activityto identify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith etal., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii); or allelicvariants and subsequences thereof (Sambrook et al., 1989, supra), asdefined herein. In a preferred aspect, the complementary strand is thefull-length complementary strand of the mature polypeptide codingsequence of SEQ ID NO: 1.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, or (iii) a full-length complementary strand of (i) or (ii); and(b) isolating the hybridizing polynucleotide, which encodes apolypeptide having cellobiohydrolase activity. In a preferred aspect,the complementary strand is the full-length complementary strand of themature polypeptide coding sequence of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137. Effective signal peptide coding sequences for filamentousfungal host cells are the signal peptide coding sequences obtained fromthe genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutralamylase, Aspergillus niger glucoamylase, Rhizomucor miehei asparticproteinase, Humicola insolens cellulase, Humicola insolens endoglucanaseV, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 17 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 51 ofSEQ ID NO: 1.

The control sequence may also be a propeptide coding sequence that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propeptide is generallyinactive and can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Myceliophthora. In amore preferred aspect, the cell is Myceliophthora thermophila. In a mostpreferred aspect, the cell is Myceliophthora thermophila CBS 202.75.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptide thatcomprises or consists of the mature polypeptide of SEQ ID NO: 2, and (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having cellobiohydrolase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, gPlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving cellobiohydrolase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellobiohydrolase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of cellobiohydrolaseactivity by fermentation of a cell that produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibitingcellobiohydrolase activity to the fermentation broth before, during, orafter the fermentation has been completed, recovering the product ofinterest from the fermentation broth, and optionally subjecting therecovered product to further purification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of cellobiohydrolaseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce thecellobiohydrolase activity substantially, and recovering the productfrom the culture broth. Alternatively, the combined pH and temperaturetreatment may be performed on an enzyme preparation recovered from theculture broth. The combined pH and temperature treatment may optionallybe used in combination with a treatment with a cellobiohydrolaseInhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the cellobiohydrolase activity. Complete removal ofcellobiohydrolase activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellobiohydrolase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thecellobiohydrolase-deficient cells may also be used to expressheterologous proteins of pharmaceutical interest such as hormones,growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellobiohydrolase activity that is produced by amethod of the present invention.

Methods of Inhibiting Expression of a Polypeptide HavingCellobiohydrolase Activity

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellobiohydrolase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in acell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencingtherapeutics. In one aspect, the invention provides methods toselectively degrade RNA using the dsRNAis of the present invention. Theprocess may be practiced in vitro, ex vivo or in viva In one aspect, thedsRNA molecules can be used to generate a loss-of-function mutation in acell, an organ or an animal. Methods for making and using dsRNAmolecules to selectively degrade RNA are well known in the art, see, forexample, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No.6,515,109; and U.S. Pat. No. 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellobiohydrolase activity of the composition has been increased, e.g.,with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having cellobiohydrolase activity, or compositions thereof,as described below.

Degradation or Conversion of Cellulosic Material

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with a composition comprising one or more cellulolytic proteinsin the presence of an effective amount of a polypeptide havingcellobiohydrolase activity of the present invention. In a preferredaspect, the method further comprises recovering the degraded orconverted cellulosic material.

The polypeptides and host cells of the present invention may be used inthe production of monosaccharides, disaccharides, and polysaccharides aschemical or fermentation feedstocks from cellulosic biomass for theproduction of ethanol, plastics, other products or intermediates. Thecomposition comprising the polypeptide having cellobiohydrolase activitymay be in the form of a crude fermentation broth with or without thecells removed or in the form of a semi-purified or purified enzymepreparation. The composition can also comprise other proteins andenzymes useful in the processing of biomass, e.g., endoglucanase,cellobiohydrolase, beta-glucosidase, hemicellulolytic enzymes, enhancers(WO 2005/074647, WO 2005/074656), etc. Alternatively, the compositionmay comprise a host cell of the present invention as a source of thepolypeptide having cellobiohydrolase activity in a fermentation processwith the biomass. In particular, the polypeptides and host cells of thepresent invention may be used to increase the value of processingresidues (dried distillers grain, spent grains from brewing, sugarcanebagasse, etc.) by partial or complete degradation of cellulose orhemicellulose. The host cell may also contain native or heterologousgenes that encode other proteins and enzymes, mentioned above, useful inthe processing of biomass.

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

In the methods of the present invention, the cellulosic material can beany material containing cellulose. Cellulose is generally found, forexample, in the stems, leaves, hulls, husks, and cobs of plants orleaves, branches, and wood of trees. The cellulosic material can be, butis not limited to, herbaceous material, agricultural residues, forestryresidues, municipal solid wastes, waste paper, and pulp and paper millresidues. The cellulosic material can be any type of biomass including,but not limited to, wood resources, municipal solid waste, wastepaper,crops, and crop residues (see, for example, Wiselogel et al., 1995, inHandbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor &Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16;Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719;Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix.

In a preferred aspect, the cellulosic material is corn stover. Inanother preferred aspect, the cellulosic material is corn fiber. Inanother preferred aspect, the cellulosic material is corn cob. Inanother preferred aspect, the cellulosic material is rice straw. Inanother preferred aspect, the cellulosic material is paper and pulpprocessing waste. In another preferred aspect, the cellulosic materialis woody or herbaceous plants. In another preferred aspect, thecellulosic material is bagasse. In another preferred aspect, thecellulosic material is orange peel.

Three major classes of enzymes are used to breakdown cellulosic biomass:

-   -   (1) The “endo-1,4-beta-glucanases” or        1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act        randomly on soluble and insoluble 1,4-beta-glucan substrates.    -   (2) The “exo-1,4-beta-D-glucanases” including both the        1,4-beta-D-glucan glucohydrolases (EC 3.2.1.74), which liberate        D-glucose from 1,4-beta-D-glucans and hydrolyze D-cellobiose        slowly, and cellobiohydrolases (1,4-beta-D-glucan        cellobiohydrolases, EC 3.2.1.91), which liberate D-cellobiose        from 1,4-beta-glucans.    -   (3) The “beta-D-glucosidases” or beta-D-glucoside        glucohydrolases (EC 3.2.1.21), which act to release D-glucose        units from cellobiose and soluble cellodextrins, as well as an        array of glycosides.

The polypeptides having cellobiohydrolase activity of the presentinvention are preferably used in conjunction with other cellulolyticproteins, e.g., endo-1,4-beta-glucanase and exo-1,4-beta-D-glucanases,to degrade the cellulose component of the biomass substrate, (see, forexample, Brigham et al., 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 119-141, Taylor & Francis, Washington D.C.; Lee,1997, Journal of Biotechnology 56: 1-24).

The endo-1,4-beta-glucanase and exo-1,4-beta-D-glucanases may beproduced by any known method known in the art (see, e.g., Bennett, J. W.and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press,CA, 1991).

The optimum amounts of a polypeptide having cellobiohydrolase activityand other cellulolytic proteins depends on several factors including,but not limited to, the mixture of component cellulolytic proteins, thecellulosic substrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation). The term “cellulolytic proteins” isdefined herein as those proteins or mixtures of proteins shown as beingcapable of hydrolyzing or converting or degrading cellulose under theconditions tested.

In a preferred aspect, the amount of polypeptide havingcellobiohydrolase activity per g of cellulosic material is about 0.5 toabout 50 mg, preferably about 0.5 to about 40 mg, more preferably about0.5 to about 25 mg, more preferably about 0.75 to about 20 mg, morepreferably about 0.75 to about 15 mg, even more preferably about 0.5 toabout 10 mg, and most preferably about 2.5 to about 10 mg per g ofcellulosic material.

In another preferred aspect, the amount of cellulolytic proteins per gof cellulosic material is about 0.5 to about 50 mg, preferably about 0.5to about 40 mg, more preferably about 0.5 to about 25 mg, morepreferably about 0.75 to about 20 mg, more preferably about 0.75 toabout 15 mg, even more preferably about 0.5 to about 10 mg, and mostpreferably about 2.5 to about 10 mg per g of cellulosic material.

In the methods of the present invention, the composition may besupplemented by one or more additional enzyme activities to improve thedegradation of the cellulosic material. Preferred additional enzymes arehemicellulases, esterases (e.g., lipases, phospholipases, and/orcutinases), proteases, laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

The enzymes may be derived or obtained from any suitable origin,including, bacterial, fungal, yeast or mammalian origin. The term“obtained” means herein that the enzyme may have been isolated from anorganism which naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism, wherein the recombinantly producedenzyme is either native or foreign to the host organism or has amodified amino acid sequence, e.g., having one or more amino acids whichare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme which is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained recombinantly, such as by site-directed mutagenesis orshuffling.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be substantially pure.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

The methods of the present invention may be used to process a cellulosicmaterial to many useful organic products, chemicals and fuels. Inaddition to ethanol, some commodity and specialty chemicals that can beproduced from cellulose include xylose, acetone, acetate, glycine,lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol,glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, cis,cis-muconic acid, and animal feed (Lynd, L. R., Wyman, C. E., andGerngross, T. U., 1999, Biocommodity Engineering, Biotechnol. Prog., 15:777-793; Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212; and Ryu, D. D. Y., andMandels, M., 1980, Cellulases: biosynthesis and applications, Enz.Microb. Technol., 2: 91-102). Potential coproduction benefits extendbeyond the synthesis of multiple organic products from fermentablecarbohydrate. Lignin-rich residues remaining after biological processingcan be converted to lignin-derived chemicals, or used for powerproduction.

Conventional methods used to process the cellulosic material inaccordance with the methods of the present invention are well understoodto those skilled in the art. The methods of the present invention may beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Such an apparatus may include a batch-stirred reactor, a continuous flowstirred reactor with ultrafiltration, a continuous plug-flow columnreactor (Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of theenzymatic hydrolysis of cellulose: 1. A mathematical model for a batchreactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor(Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose byusing an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or areactor with intensive stirring induced by an electromagnetic field(Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y.,Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis usinga novel type of bioreactor with intensive stirring induced byelectromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol is combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews 66: 506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,malonic acid, oxalic acid, propionic acid, succinic acid, and xylonicacid); ketones (e.g., acetone); amino acids (e.g., aspartic acid,glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

The present invention further relates to methods of producing asubstance, comprising: (a) saccharifying a cellulosic material with aneffective amount of a composition comprising one or more cellulolyticproteins in the presence of an effective amount of a polypeptide havingcellobiohydrolase activity of the present invention; (b) fermenting thesaccharified cellulosic material of step (a) with one or morefermentating microorganisms; and (c) recovering the substance from thefermentation. The composition comprising the polypeptide havingcellobiohydrolase activity may be in the form of a crude fermentationbroth with or without the cells removed or in the form of asemi-purified or purified enzyme preparation or the composition maycomprise a host cell of the present invention as a source of thepolypeptide having cellobiohydrolase activity in a fermentation processwith the biomass.

The substance can be any substance derived from the fermentation. In apreferred embodiment, the substance is an alcohol. It will be understoodthat the term “alcohol” encompasses a substance that contains one ormore hydroxyl moieties. In a more preferred embodiment, the alcohol isarabinitol. In another more preferred embodiment, the alcohol isbutanol. In another more preferred embodiment, the alcohol is ethanol.In another more preferred embodiment, the alcohol is glycerol. Inanother more preferred embodiment, the alcohol is methanol. In anothermore preferred embodiment, the alcohol is 1,3-propanediol. In anothermore preferred embodiment, the alcohol is sorbitol. In another morepreferred embodiment, the alcohol is xylitol. See, for example, Gong, C.S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred embodiment, the substance is an organic acid. Inanother more preferred embodiment, the organic acid is acetic acid. Inanother more preferred embodiment, the organic acid is acetonic acid. Inanother more preferred embodiment, the organic acid is adipic acid. Inanother more preferred embodiment, the organic acid is ascorbic acid. Inanother more preferred embodiment, the organic acid is citric acid. Inanother more preferred embodiment, the organic acid is2,5-diketo-D-gluconic acid. In another more preferred embodiment, theorganic acid is formic acid. In another more preferred embodiment, theorganic acid is fumaric acid. In another more preferred embodiment, theorganic acid is glucaric acid. In another more preferred embodiment, theorganic acid is gluconic acid. In another more preferred embodiment, theorganic acid is glucuronic acid. In another more preferred embodiment,the organic acid is glutaric acid. In another preferred embodiment, theorganic acid is 3-hydroxypropionic acid. In another more preferredembodiment, the organic acid is itaconic acid. In another more preferredembodiment, the organic acid is lactic acid. In another more preferredembodiment, the organic acid is malic acid. In another more preferredembodiment, the organic acid is malonic acid. In another more preferredembodiment, the organic acid is oxalic acid. In another more preferredembodiment, the organic acid is propionic acid. In another morepreferred embodiment, the organic acid is succinic acid. In another morepreferred embodiment, the organic acid is xylonic acid. See, forexample, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another preferred embodiment, the substance is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. In another more preferred embodiment, theketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

In another preferred embodiment, the substance is an amino acid. Inanother more preferred embodiment, the organic acid is aspartic acid. Inanother more preferred embodiment, the amino acid is glutamic acid. Inanother more preferred embodiment, the amino acid is glycine. In anothermore preferred embodiment, the amino acid is lysine. In another morepreferred embodiment, the amino acid is serine. In another morepreferred embodiment, the amino acid is threonine. See, for example,Richard, A., and Margaritis, A., 2004, Empirical modeling of batchfermentation kinetics for poly(glutamic acid) production and othermicrobial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred embodiment, the substance is a gas. In another morepreferred embodiment, the gas is methane. In another more preferredembodiment, the gas is H₂. In another more preferred embodiment, the gasis CO₂. In another more preferred embodiment, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Production of a substance from cellulosic material typically requiresfour major steps. These four steps are pretreatment, enzymatichydrolysis, fermentation, and recovery. Exemplified below is a processfor producing ethanol, but it will be understood that similar processescan be used to produce other substances, for example, the substancesdescribed above.

Pretreatment.

In the pretreatment or pre-hydrolysis step, the cellulosic material isheated to break down the lignin and carbohydrate structure, solubilizemost of the hemicellulose, and make the cellulose fraction accessible tocellulolytic enzymes. The heating is performed either directly withsteam or in slurry where a catalyst may also be added to the material tospeed up the reactions. Catalysts include strong acids, such as sulfuricacid and SO₂, or alkali, such as sodium hydroxide. The purpose of thepre-treatment stage is to facilitate the penetration of the enzymes andmicroorganisms. Cellulosic biomass may also be subject to a hydrothermalsteam explosion pre-treatment (See U.S. Patent Application No.20020164730).

Saccharification.

In the enzymatic hydrolysis step, also known as saccharification,enzymes as described herein are added to the pretreated material toconvert the cellulose fraction to glucose and/or other sugars. Thesaccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions. Asaccharification step may last up to 200 hours. Saccharification may becarried out at temperatures from about 30° C. to about 65° C., inparticular around 50° C., and at a pH in the range between about 4 andabout 5, especially around pH 4.5. To produce glucose that can bemetabolized by yeast, the hydrolysis is typically performed in thepresence of a beta-glucosidase.

Fermentation.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to ethanol by a fermenting organism, such as yeast. Thefermentation can also be carried out simultaneously with the enzymatichydrolysis in the same vessel, again under controlled pH, temperature,and mixing conditions. When saccharification and fermentation areperformed simultaneously in the same vessel, the process is generallytermed simultaneous saccharification and fermentation or SSF.

Any suitable cellulosic substrate or raw material may be used in afermentation process of the present invention. The substrate isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention include cellulose-containingmaterials, such as wood or plant residues or low molecular sugars DP1-3obtained from processed cellulosic material that can be metabolized bythe fermenting microorganism, and which may be supplied by directaddition to the fermentation medium.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsare able to ferment, i.e., convert, sugars, such as glucose, xylose,arabinose, mannose, galactose, or oligosaccharides directly orindirectly into the desired fermentation product. Examples of fermentingmicroorganisms include fungal organisms, such as yeast. Preferred yeastincludes strains of the Saccharomyces spp., and in particular,Saccharomyces cerevisiae. Commercially available yeast include, e.g.,RED STAR®/Lesaffre Ethanol Red (available from Red Star/Lesaffre, USA)FALI (available from Fleischmann's Yeast, a division of Burns Philp FoodInc., USA), SUPERSTART® (available from Alltech), GERT STRAND®(available from Gert Strand AB, Sweden) and FERMIOL® (available from DSMSpecialties).

In a preferred embodiment, the yeast is a Saccharomyces spp. In a morepreferred embodiment, the yeast is Saccharomyces cerevisiae. In anothermore preferred embodiment, the yeast is Saccharomyces distaticus. Inanother more preferred embodiment, the yeast is Saccharomyces uvarum. Inanother preferred embodiment, the yeast is a Kluyveromyces. In anothermore preferred embodiment, the yeast is Kluyveromyces marxianus. Inanother more preferred embodiment, the yeast is Kluyveromyces fragilis.In another preferred embodiment, the yeast is a Candida. In another morepreferred embodiment, the yeast is Candida pseudotropicalis. In anothermore preferred embodiment, the yeast is Candida brassicae. In anotherpreferred embodiment, the yeast is a Clavispora. In another morepreferred embodiment, the yeast is Clavispora lusitaniae. In anothermore preferred embodiment, the yeast is Clavispora opuntiae. In anotherpreferred embodiment, the yeast is a Pachysolen. In another morepreferred embodiment, the yeast is Pachysolen tannophilus. In anotherpreferred embodiment, the yeast is a Bretannomyces. In another morepreferred embodiment, the yeast is Bretannomyces clausenii (Philippidis,G. P., 1996, Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z.,Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho, N. W. Y., Chen, Z, Brainard, A.P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol. 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or another microorganism typically is added to the degradedcellulose or hydrolysate and the fermentation is ongoing for about 24 toabout 96 hours, such as about 35 to about 60 hours. The temperature istypically between about 26° C. to about 40° C., in particular at about32° C., and at about pH 3 to about pH 6, in particular around pH 4-5.

In a preferred embodiment, yeast or another microorganism is applied tothe degraded cellulose or hydrolysate and the fermentation is ongoingfor about 24 to about 96 hours, such as typically 35-60 hours. In apreferred embodiments, the temperature is generally between about 26 toabout 40° C., in particular about 32° C., and the pH is generally fromabout pH 3 to about pH 6, preferably around pH 4-5. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 5×10⁷ viable count per ml of fermentation broth. During anethanol producing phase the yeast cell count should preferably be in therange from approximately 10⁷ to 10¹⁰, especially around approximately2×10⁸. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process, Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients comprisingP, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery.

The alcohol is separated from the fermented cellulosic material andpurified by conventional methods of distillation. Ethanol with a purityof up to about 96 vol. % ethanol can be obtained, which can be used as,for example, fuel ethanol, drinking ethanol, i.e., potable neutralspirits, or industrial ethanol.

For other substances, any method known in the art can be used including,but not limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing), differentialsolubility (e.g., ammonium sulfate precipitation), SDS-PAGE,distillation, or extraction.

In the methods of the present invention, the polypeptide havingcellobiohydrolase activity and other cellulolytic protein(s) may besupplemented by one or more additional enzyme activities to improve thedegradation of the cellulosic material. Preferred additional enzymes arehemicellulases, esterases (e.g., lipases, phospholipases, and/orcutinases), proteases, laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

Detergent Compositions

The present invention also relates to detergent compositions, comprisinga surfactant and a polypeptide having cellobiohydrolase activity of thepresent invention. The polypeptides having cellobiohydrolase activitymay be added to and thus become a component of a detergent composition.

The detergent composition of the present invention may be, for example,formulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or formulated asa detergent composition for use in general household hard surfacecleaning operations, or formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising the polypeptides having cellobiohydrolase activityof the present invention. The detergent additive as well as thedetergent composition may comprise one or more other enzymes such as aprotease, lipase, cutinase, an amylase, carbohydrase, cellulase,pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., alaccase, and/or peroxidase.

In general the properties of the enzymatic components should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzymatic components should be present in effective amounts.

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include ALCALASE™,SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novozymes A/S),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131, 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipases include LIPOLASE™, LIPEX™, andLIPOLASE ULTRA™ (Novozymes A/S).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Amylases include, for example, α-amylases obtained from Bacillus, e.g.,a special strain of Bacillus licheniformis, described in more detail inGB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ andBAN™ (Novozymes A/S), RAPIDASE™ and PURASTAR™ (from GenencorInternational Inc.).

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include CELLUZYME™, and CAREZYME™(Novozymes A/S), CLAZINASE™, and PURADAX HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ (Novozymes A/S).

The enzymatic component(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the present invention, i.e., a separate additive or a combinedadditive, can be formulated, for example, as a granulate, liquid,slurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the present invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system that may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzymatic component(s) of the detergent composition of the presentinvention may be stabilized using conventional stabilizing agents, e.g.,a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol,lactic acid, boric acid, or a boric acid derivative, e.g., an aromaticborate ester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid, and the composition may be formulated as described in, forexample, WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzymatic component, in particular thepolypeptides having cellobiohydrolase activity of the present invention,may be added in an amount corresponding to 0.01-100 mg of enzyme proteinper liter of wash liquor, preferably 0.05-5 mg of enzyme protein perliter of wash liquor, in particular 0.1-1 mg of enzyme protein per literof wash liquor.

The polypeptides having cellobiohydrolase activity of the presentinvention may additionally be incorporated in the detergent formulationsdisclosed in WO 97/07202 which is hereby incorporated as reference.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to anucleotide sequence encoding a signal peptide comprising or consistingof amino acids 1 to 17 of SEQ ID NO: 2, wherein the gene is foreign tothe nucleotide sequence.

In a preferred aspect, the nucleotide sequence comprises or consists ofnucleotides 1 to 51 of SEQ ID NO: 1.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods of producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides that comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more (several) may be heterologous or native to the hostcell. Proteins further include naturally occurring allelic andengineered variations of the above mentioned proteins and hybridproteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Myceliophthora thermophila CBS 202.75 was used as the source of the genefor a Family 6 polypeptide having cellobiohydrolase activity.Aspergillus oryzae JaL355 strain (WO 2002/40694) was used for expressionof the Myceliophthora thermophila gene encoding the polypeptide havingcellobiohydrolase activity.

Media

Minimal medium plates were composed per liter of 6 g of NaNO₃, 0.52 g ofKCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g ofNoble agar, 20 ml of 50% glucose, 2.5 ml of MgSO₄.7H₂O, and 20 ml of a0.02% biotin solution.

COVE trace elements solution was composed per liter of 0.04 g ofNa₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, and 0.5 ml of AMG trace metals solution; pH 5.0.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.7H₂O, and 3 g of citric acid.

YEG medium was composed per liter of 20 g of dextrose and 5 g of yeastextract.

Example 1 Myceliophthora thermophila CBS 202.75 Genomic DNA Extraction

Myceliophthora thermophila CBS 202.75 was grown in 100 ml of YEG mediumin a baffled shake flask at 45° C. and 200 rpm for 2 days. Mycelia wereharvested by filtration using MIRACLOTH® (Calbiochem, La Jolla, Calif.,USA), washed twice in deionized water, and frozen under liquid nitrogen.Frozen mycelia were ground, by mortar and pestle, to a fine powder, andtotal DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc.,Valencia, Calif., USA).

Example 2 Isolation of a Full-Length Family 6 Cellobiohydrolase Gene(cel6a) from Myceliophthora thermophila CBS 202.75

A full-length Family 6 cellobiohydrolase gene (cel6a) was isolated fromMyceliophthora thermophila CBS 202.75 using a GENOMEWALKER™ UniversalKit (Clontech Laboratories, Inc., Mountain View, Calif., USA) accordingto the manufacturer's instructions. Briefly, total genomic DNA fromMyceliophthora thermophila CBS 202.75 was digested separately with fourdifferent restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) thatleave blunt ends. Each batch of digested genomic DNA was then ligatedseparately to the GENOMEWALKER™ Adaptor (Clontech Laboratories, Inc.,Mountain View, Calif., USA) to create four libraries. These librarieswere then employed as templates in PCR reactions using two gene-specificprimers shown below, one for primary PCR and one for secondary PCR. Theprimers were designed based on a partial Family 6 cellobiohydrolase gene(cel6a) sequence from Myceliophthora thermophila (WO 2004/056981).

Primer MtCel6a-R4: (SEQ ID NO: 3) 5′-ATTGGCAGCCCGGATCTGGGACAGAGTCTG-3′Pimer MtCel6a-R5: (SEQ ID NO: 4) 5′-CCGGTCATGCTAGGAATGGCGAGATTGTGG-3′

The primary amplifications were composed of 1 μl (approximately 6 ng) ofeach library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10pmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View,Calif., USA), 10 pmol of primer MtCel6a-R4, 2 μl of 1× ADVANTAGE®GC-Melt LA Buffer (Clontech Laboratories, Inc., Mountain View, Calif.,USA), and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix (ClontechLaboratories, Inc., Mountain View, Calif., USA) in a final volume of 25μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER®5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed forpre-denaturing at 94° C. for 1 minute; seven cycles each at a denaturingtemperature of 94° C. for 30 seconds; annealing and elongation at 72° C.for 5 minutes; and 32 cycles each at 67° C. for 5 minutes.

The secondary ampliifications were composed of 1 μl of each primary PCRproduct as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmolof Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif.,USA), 10 pmol of primer MtCel6a-R5, 1× ADVANTAGE® GC-Melt LA Buffer, and1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of25 μl. The amplifications were performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute;5 cycles each at a denaturing temperature of 94° C. for 30 seconds;annealing and elongation at 72° C. for 5 minutes; and 20 cycles at 67°C. for 5 minutes.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE)buffer where a 3.5 kb product band from the Eco RV library was excisedfrom the gel, purified using a QIAQUICK® Gel Extraction Kit (QIAGEN,Valencia, Calif., USA) according to the manufacturer's instructions, andsequenced.

Example 3 Characterization of the Myceliophthora thermophila GenomicSequence Encoding a Family 6 Cellobiohydrolase

DNA sequencing of the 3.5 kb PCR fragment was performed with aPerkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. The following genespecific primers were used for sequencing:

MtCel6a-F2: (SEQ ID NO: 5) 5′-GCTGTAAACTGCGAATGGGTTCAG-3′ MtCel6a-F3:(SEQ ID NO: 6) 5′-GGGTCCCACATGCTGCGCCT-3′ MtCel6a-R8: (SEQ ID NO: 7)5′-AAAATTCACGAGACGCCGGG-3′

Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The 3.5 kb sequence wascompared and aligned with a partial Family 6 cellobiohydrolase gene(cel6a) sequence from Myceliophthora thermophila (WO 2004/056981).

A gene model for the Myceliophthora thermophila sequence was constructedbased on similarity of the encoded protein to homologous glycosidehydrolase Family 6 proteins from Thielavia terrestris, Chaetomiumthermophilum, Humicola insolens, and Trichoderma reesei. The nucleotidesequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2)are shown in FIGS. 1A and 1B. The genomic fragment encodes a polypeptideof 482 amino acids, interrupted by 3 introns of 96, 87, and 180 bp. The% G+C content of the gene and the mature coding sequence are 61.6% and64%, respectively. Using the SignalP software program (Nielsen et al.,1997, Protein Engineering 10:1-6), a signal peptide of 17 residues waspredicted. The predicted mature protein contains 465 amino acids with amolecular mass of 49.3 kDa.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Myceliophthora thermophila gene encoding the CEL6Amature polypeptide having cellobiohydrolase activity shared 78.6% and77.6% identity (excluding gaps) to the deduced amino acid sequences oftwo glycoside hydrolase Family 6 proteins from Chaetomium thermophilumand Humicola insolens, respectively (GeneSeqP accession numbers ADP84824and AAW44853, respectively).

Example 4 Cloning of the Myceliophthora thermophila CellobiohydrolaseGene (Cel6a) and Construction of an Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Myceliophthora thermophila cellobiohydrolase gene from thegenomic DNA prepared in Example 1. An InFusion Cloning Kit (BDBiosciences, Palo Alto, Calif., USA) was used to clone the fragmentdirectly into the expression vector pAlLo2 (WO 2004/099228), without theneed for restriction digestion and ligation.

MtCel6a-F4: (SEQ ID NO: 8) 5′-ACTGGATTTACCATGGCCAAGAAGCTTTTCATCACC-3′MtCel6a-R9: (SEQ ID NO: 9) 5′-TCACCTCTAGTTAATTAATTAGAAGGGCGGGTTGGCGT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pAlLo2.

Fifty picomoles of each of the primers above were used in a PCR reactioncomposed of 100 ng of Myceliophthora thermophila genomic DNA, 2 μl of 1×ADVANTAGE® GC-Melt LA Buffer, 0.4 mM each of dATP, dTTP, dGTP, and dCTP,and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volumeof 25 μl. The amplification was performed using an EPPENDORF®MASTERCYCLER® 5333 programmed for 1 cycle at 94° C. for 1 minutes; and30 cycles each at 94° C. for 30 seconds, 62° C. for 30 seconds, and 72°C. for 2 minutes. The heat block then went to a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1842 bp product band was excised from the gel,and purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

Plasmid pAlLo2 (WO 2004/099228) was digested with Nco I and Pac I,isolated by 1.0% agarose gel electrophoresis using TAE buffer, andpurified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anInfusion Cloning Kit resulting in pSMai180 (FIG. 2) in whichtranscription of the cellobiohydrolase gene was under the control of ahybrid of promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase (NA2-tpipromoter). The ligation reaction (50 μl) was composed of 1× InFusionBuffer (BD Biosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences,Palo Alto, Calif., USA), 1 μl of Infusion enzyme (diluted 1:10) (BDBiosciences, Palo Alto, Calif., USA), 100 ng of pAlLo2 digested with NcoI and Pac I, and 50 ng of the Myceliophthora thermophila cel6a purifiedPCR product. The reaction was incubated at room temperature for 30minutes. One μl of the reaction was used to transform E. coli XL10SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, Calif., USA).An E. coli transformant containing pSMai180 was detected by restrictiondigestion and plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGENInc., Valencia, Calif., USA). The Myceliophthora thermophila cel6ainsert in pSMai180 was confirmed by DNA sequencing.

The same 1842 bp PCR fragment was cloned into pCR02.1—TOPO vector(Invitrogen, Carlsbad, Calif., USA) using a TOPO TA CLONING® Kit, togenerate pSMai182 (FIG. 2). The Myceliophthora thermophila cel6a insertin pSMai182 was confirmed by DNA sequencing. E. coli pSMai182 wasdeposited with the Agricultural Research Service Patent CultureCollection, Northern Regional Research Center, Peoria, Ill., USA, onSep. 6, 2007.

Example 6 Expression of the Myceliophthora thermophila Family 6Cellobiohydrolase cel6a Gene in Aspergillus oryzae JaL355

Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Three μg of pSMai180 were used to transform Aspergillusoryzae JaL355.

The transformation of Aspergillus oryzae JaL355 with pSMai180 yieldedabout 50 transformants. Twenty transformants were isolated to individualMinimal medium plates.

Confluent Minimal Medium plates of the 20 transformants were washed with5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.After 5 days incubation, 5 μl of supernatant from each culture wereanalyzed on CRITERION® Tris-HCl gels (Bio-Rad Laboratories, Hercules,Calif., USA) with a CRITERION® Cell (Bio-Rad Laboratories, Hercules,Calif., USA), according to the manufacturer's instructions. Theresulting gel was stained with BIO-SAFE™ Coomassie Stain (Bio-RadLaboratories, Hercules, Calif., USA). SDS-PAGE profiles of the culturesshowed that the majority of the transformants had a major band ofapproximately 70 kDa.

A confluent plate of one transformant, designated transformant 14, waswashed with 10 ml of 0.01% TWEEN® 20 and inoculated into a 2 literFernbach containing 500 ml of MDU2BP medium to generate broth forcharacterization of the enzyme. The culture was harvested on day 5 andfiltered using a 0.22 μm EXPRESS™ Plus Membrane (Millipore, Bedford,Mass., USA).

Example 7 Characterization of Myceliophthora thermophila CEL6ACellobiohydrolase

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL), Boulder, Colo., USA, using dilutesulfuric acid. The following conditions were used for the pretreatment:0.048 g sulfuric acid per g dry biomass at 190° C. and 25% w/w drysolids for around 1 minute. The water-insoluble solids in the pretreatedcorn stover (PCS) contained 53.2% cellulose, 3.6% hemicellulose and29.8% lignin. Cellulose and hemicellulose were determined by a two-stagesulfuric acid hydrolysis with subsequent analysis of sugars by highperformance liquid chromatography using NREL Standard AnalyticalProcedure #002. Lignin was determined gravimetrically after hydrolyzingthe cellulose and hemicellulose fractions with sulfuric acid using NRELStandard Analytical Procedure #003. Prior to enzymatic hydrolysis, thePCS was washed with a large volume of distilled water until the pH washigher than 4.0, then sieved through 100-mash sieve, and finallyautoclaved at 121° C. for 30 minutes. The dry content of the washed andsieved PCS was found to be 6.54%.

Shake flask broth of Myceliophthora thermophila CEL6A cellobiohydrolaseprepared as described in Example 6 was desalted using an ECONO-PAC® 10DGcolumn (Bio-Rad Laboratories, Inc., Hercules, Calif., USA). Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kit(Pierce, Rockford, Ill., USA).

Trichoderma reesei CEL7B endoglucanase I was cloned and expressed inAspergillus oryzae JaL250 as described in WO 2005/067531. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kit.The Trichoderma reesei CEL7B endoglucanase I was desalted and bufferexchanged in 150 mM NaCl-20 mM sodium acetate pH 5.0 using a HIPREP®26/10 Desalting Column (GE Healthcare Life Sciences, Piscataway, N.J.,USA) according to the manufacturer's instructions.

Aspergillus oryzae Cel3A beta-glucosidase was recombinantly prepared asdescribed in WO 2004/099228, and purified as described by Langston etal., 2006, Biochim. Biophys. Acta Proteins Proteomics 1764: 972-978.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit. Penicillium brasilianum IBT 20888 Cel3A beta-glucosidase wasrecombinantly produced and desalted using a HIPREP® 26/10 DesaltingColumn, as above, and further purified with a Mono Q® column using anAKTA FPLC System (GE Healthcare Life Sciences, Piscataway, N.J., USA)according to the manufacturer's instructions.

Trichoderma reesei CEL6A cellobiohydrolase gene was isolated fromTrichoderma reesei RutC30 as described in WO 2005/056772. TheTrichoderma reesei CEL6A cellobiohydrolase gene was expressed inFusarium venenatum using pEJG61 as an expression vector according to theprocedures described in U.S. Patent Application 20060156437.Fermentation was performed as described in U.S. Patent Application20060156437. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit. Trichoderma reesei CEL6A cellobiohydrolase wasdesalted and buffer-exchanged into 20 mM sodium acetate-150 mM NaCl pH5.0 using a HIPREP® 26/10 Desalting column according to themanufacturer's instructions.

Hydrolysis of PCS was performed in 96-deep-well plates (AxygenScientific, Union City, Calif., USA) sealed by a plate sealer (ALPS-300,Abgene, Epsom, UK), with a total reaction volume of 1.0 ml. To test theactivity of Myceliophthora thermophila cellobiohydrolase, PCS was loadedat 1 mg of cellobiohydrolase per g cellulose, together with 0.5 mg ofAspergillus oryzae beta-glucosidase per g cellulose. Trichoderma reeseiCEL6A cellobiohydrolase (1 mg per g cellulose) together with Aspergillusoryzae beta-glucosidase (0.5 mg per g cellulose) were used as a controlfor comparison to the Myceliophthora thermophila CEL6Acellobiohydrolase. PCS hydrolysis was performed at pH 5.0, 50° C. in aTS Autoflow CO₂ Jacketed Incubator (Fisher Scientific, Pittsburgh, Pa.,USA). Reactions were run in triplicates and aliquots were taken duringthe course of hydrolysis. PCS hydrolysis reactions were stopped bymixing a 20 μl aliquot of each hydrolyzate with 180 μl of 0.1 M NaOH(stop reagent). Appropriate serial dilutions were generated for eachsample and the reducing sugar content determined using apara-hydroxybenzoic acid hydrazide (PHBAH, Sigma, St. Louis, Mo., USA)assay adapted to a 96 well microplate format. A 100 μl aliquot of anappropriately diluted sample was placed in a 96 well conical bottomedmicroplate. Reactions were initiated by adding 50 μl of 1.5% (w/v) PHBAHin 2% NaOH to each well. Plates were heated uncovered at 95° C. for 10minutes, after which 50 μl of distilled water was added to each well. A100 μl aliquot from each well was transferred to a flat bottomed 96 wellplate and the absorbance at 410 nm was measured using a SPECTRAMAX®Microplate Reader (Molecular Devices, Sunnyvale, Calif., USA). Glucosestandards (0.1-0.0125 mg/ml diluted with 0.4% sodium hydroxide) wereused to prepare a standard curve to translate the obtained A_(410 nm)values into glucose equivalents. The resultant equivalents were used tocalculate the percentage of PCS cellulose conversion for each reaction.

The degree of cellulose conversion to reducing sugar (% conversion) wascalculated using the following equation:

%Conversion=RS_((mg/ml))×100×162/(cellulose_((mg/ml))×180)==RS_((mg/ml))×100/(cellulose_((mg/ml))×1.111)

In this equation, RS is the concentration of reducing sugar in solutionmeasured in glucose equivalents (mg/ml), and the factor 1.111 reflectsthe weight gain in converting cellulose to glucose.

The results are summarized in Table 1. Myceliophthora thermophila CEL6Acellobiohydrolase had similar or slightly higher activity on PCS thanTrichoderma reesei CEL6A cellobiohydrolase in the presence ofAspergillus oryzae beta-glucosidase.

TABLE 1 Cellulose conversion by Myceliophthora thermophila CEL6Acellobiohydrolase or Trichoderma reesei CEL6A cellobiohydrolase plusAspergillus oryzae beta-glucosidase Conversion Test Loading, at 65hours, # Enzyme Name mg/g cellulose % 1 M. thermophila CBH + A. oryzae1 + 0.5 5.0 beta-glucosidase 2 T. reesei CBH + A. oryzae 1 + 0.5 3.7beta-glucosidase

Desalted Myceliophthora thermophila Cel6A was further purified through aPHENYL SUPEROSE® HR 16/10 column (GE Healthcare Life Sciences,Piscataway, N.J., USA) using an AKTA FPLC System. SDS-PAGE of the sampleshowed that it was at least 90% pure. Other enzymes used below were alsofurther purified, as above, using an AKTA FPLC System, with purityhigher than 90%.

Hydrolysis of PCS (40 g/L in reaction) was conducted in 96-deep-wellplates and sealed as described above, with a total reaction volume of1.0 ml. Myceliophthora thermophila Cel6A cellobiohydrolase, incomparison with Trichoderma reesei Cel6A cellobiohydrolase, were testedin PCS hydrolysis using artificial enzyme mixtures at pH 5, 50 and 55°C. (TS Autoflow CO₂ Jacketed Incubator). The enzyme mixtures werecomposed of Trichoderma reesei Cel7B endoglucanase (2 mg/g cellulose),Penicillium brasilianum GH3A beta-glucosidase (0.3 mg/g cellulose), andeither Myceliophthora thermophila Cel6A cellobiohydrolase or Trichodermareesei Cel6A cellobiohydrolase, (2 mg/g cellulose). PCS hydrolysisreactions were stopped by mixing a 20 μl aliquot of each hydrolyzatewith 180 μl of 0.1 M NaOH (stop reagent). Analysis of the hydrolysisreactions and calculations were performed as described above. Celluloseconversion results are summarized in Table 2.

TABLE 2 Cellulose conversion by Trichoderma reesei Cel7B endoglucanase(2 mg/g cellulose), Penicillium brasilianum GH3A beta-glucosidase (0.3mg/g cellulose), and either Myceliophthora thermophila Cel6A orTrichoderma reesei Cel6A cellobiohydrolase (2 mg/g cellulose), pH 5, 50and 55° C. Conversion Test Enzyme Name Temperature, at 72 h, # (mg/gcellulose) ° C. % 1 T. reesei Cel7B endoglucanase (2) + 50 22.5 P.brasilianum GH3A beta-glucosidase (0.3) + M. thermophila Cel6Acellobiohydrolase (2) 2 T. reesei Cel7B endoglucanase (2) + 50 19.6 P.brasilianum GH3A beta-glucosidase (0.3) + T. reesei Cel6Acellobiohydrolase (2) 3 T. reesei Cel7B endoglucanase (2) + 55 25.9 P.brasilianum GH3A beta-glucosidase (0.3) + M. thermophila Cel6Acellobiohydrolase (2) 4 T. reesei Cel7B endoglucanase (2) + 55 18.2 P.brasilianum GH3A (0.3) + T. reesei Cel6A cellobiohydrolase (2)

Table 1 showed that the Myceliophthora thermophila 6A cellobiohydrolaseoutperformed the Trichoderma reesei 6A cellobiohydrolase in PCShydrolysis at both 50 and 55° C., and especially at 55° C.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., USA, and given the following accessionnumber:

Deposit Accession Number Date of Deposit E. coli pSMai182 NRRL B-50059Sep. 6, 2007

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A method of producing a mutant cell, comprising disrupting ordeleting a nucleotide sequence encoding a polypeptide havingcellobiohydrolase activity in a parent cell, which results in the mutantproducing less of the polypeptide than the parent cell, wherein thepolypeptide having cellobiohydrolase activity is selected from the groupconsisting of: (a) a polypeptide having cellobiohydrolase activitycomprising an amino acid sequence having at least 99% identity to aminoacids 18 to 842 of SEQ ID NO: 2; and (b) a polypeptide havingcellobiohydrolase activity comprising amino acids 18 to 482 of SEQ IDNO:
 2. 2. The method of claim 1, wherein the polypeptide is encoded bythe polynucleotide contained in plasmid pSMai182 which is contained inE. coli NRRL B-50059.
 3. The method of claim 1, wherein the mutant cellis a Myceliophthora cell.
 4. The method of claim 3, wherein theMyceliophthora cell is a Myceliophthora thermophila cell.
 5. A mutantcell, comprising a disruption or deletion of a nucleotide sequenceencoding a polypeptide having cellobiohydrolase activity in a parentcell, wherein the mutant produces less of the polypeptide than theparent cell, and wherein the polypeptide having cellobiohydrolaseactivity is selected from the group consisting of: (a) a polypeptidehaving cellobiohydrolase activity comprising an amino acid sequencehaving at least 99% identity to amino acids 18 to 842 of SEQ ID NO: 2;and (b) a polypeptide having cellobiohydrolase activity comprising aminoacids 18 to 482 of SEQ ID NO:
 2. 6. The mutant cell of claim 5, whereinthe polypeptide is encoded by the polynucleotide contained in plasmidpSMai182 which is contained in E. coli NRRL B-50059.
 7. The mutant cellof claim 5, further comprising a gene encoding a protein.
 8. The mutantcell of claim 7, wherein the protein is a native protein.
 9. The mutantcell of claim 7, wherein the protein is a heterologous protein.
 10. Themutant cell of claim 5, wherein the mutant cell is a Myceliophthoracell.
 11. The mutant cell of claim 10, wherein the Myceliophthora cellis a Myceliophthora thermophila cell.
 12. A method of producing aprotein, comprising: (a) cultivating the mutant cell of claim 7 underconditions conducive for production of the protein; and (b) recoveringthe protein.
 13. The method of claim 12, wherein the mutant cell is aMyceliophthora cell.
 14. The method of claim 13, wherein theMyceliophthora cell is a Myceliophthora thermophila cell.